Infections by human rhinovirus (HRV) contribute to a diversity of human illnesses that include the common cold, bronchitis, exacerbation of asthma and other chronic lung diseases. Patient biopsies of HRV-infected tissues from the upper and lower airways reveal patches of robust infection dispersed among areas of healthy tissue. We hypothesize that differing tissue susceptibility to HRV infection reflects an underlying and intrinsic cell-level heterogeneity in responses to virus infection and antiviral immune signals. Testing this hypothesis poses a number of challenges: standard measures of cell behavior sampled from large cell populations provide an average, not single-cell measures; spreading infections are by definition complex and heterogeneous environments; and most mechanistic studies of viral growth and infection spread lack host factors that potentially promote virus spread. To address these challenges we will build on our significant experience measuring single-cell infections, as well as implementing and quantifying infection spread in controlled cell-culture environments. We propose to employ: (i) a dual-color fluorescent reporter method to provide simultaneous readouts of viral and cellular gene expression from HRV-infected cells, (ii) a quantitative imaging acquisition and analysis platform for high-throughput measures of dynamic single-cell behaviors, and (iii) a micro-scale cell-culture technology that offers unprecedented environmental control over cell density, localized virus inoculation, and flow conditions. Preliminary results indicate the feasibility of these technologies and significant opportunity for elucidating mechanisms of viral and cellular gene expression, virus growth, and infection spread. Our project aims to: (1) advance a data-driven integrated understanding of HRV single-cycle growth in cell cultures, (2) elucidate the impact of cell-virus interactions on HRV infection spread, and (3) isolate, characterize and utilize host factors that promote growth and spread of HRV in vivo. These aims will make extensive use of molecular and clinical tools in the other two projects, including airway epithelial cells and chimeric viruses. Results from this project will provide novel insights into how some tissues resist infection while others are susceptible, and this information will guide the development of improved treatment strategies for HRV-related illnesses.